Seed oil content has traditionally been modified by plant breeding. The use of recombinant DNA technology to alter seed oil composition can accelerate this process and in some cases alter seed oils in a way that cannot be accomplished by breeding alone. The oil composition of Brassica has been significantly altered by modifying the expression of a number of lipid metabolism genes. Such manipulations of seed oil composition have focused on altering the proportion of endogenous component fatty acids. For example, antisense repression of the .DELTA.12-desaturase gene in transgenic rapeseed has resulted in an increase in oleic acid of up to 83%. Topfer et al. 1995 Science 268:681-686.
There have been some successful attempts at modifying the composition of seed oil in transgenic plants by introducing new genes that allow the production of a fatty acid that the host plants were not previously capable of synthesizing. Van de Loo, et al. (1995 Proc. Natl. Acad. Sci USA 92:6743-6747) have been able to introduce a .DELTA.12-hydroxylase gene into transgenic tobacco, resulting in the introduction of a novel fatty acid, ricinoleic acid, into its seed oil. The reported accumulation was modest from plants carrying constructs in which transcription of the hydroxylase gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Similarly, tobacco plants have been engineered to produce low levels of petroselinic acid by expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992 Proc. Natl. Acad. Sci USA 89:11184-11188).
The long chain fatty acids (C18 and larger), have significant economic value both as nutritionally and medically important foods and as industrial commodities (Ohlrogge, J. B. 1994 Plant Physiol. 104:821-826). Linoleic (18:2 .DELTA.9,12) and .alpha.-linolenic acid (18:3 .DELTA.9,12,15) are essential fatty acids found in many seed oils. The levels of these fatty-acids have been manipulated in oil seed crops through breeding and biotechnology (Ohlrogge, et al. 1991 Biochim. Biophys. Acta 1082:1-26; Topfer et al. 1995 Science 268:681-686). Additionally, the production of novel fatty acids in seed oils can be of considerable use in both human health and industrial applications.
Consumption of plant oils rich in .gamma.-linolenic acid (GLA) (18:3 .DELTA.6,9,12) is thought to alleviate hypercholesterolemia and other related clinical disorders which correlate with susceptibility to coronary heart disease (Brenner R. R. 1976 Adv. Exp. Med. Biol. 83:85-101). The therapeutic benefits of dietary GLA may result from its role as a precursor to prostaglandin synthesis (Weete, J. D. 1980 in Lipid Biochemistry of Fungi and Other Organisms, eds. Plenum Press, New York, pp. 59-62). Linoleic acid(18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme .DELTA.6-desaturase.
Few seed oils contain GLA despite high contents of the precursor linoleic acid. This is due to the absence of .DELTA.6-desaturase activity in most plants. For example, only borage (Borago officinalis), evening primrose (Oenothera biennis), and currants (Ribes nigrum) produce appreciable amounts of linolenic acid. Of these three species, only Oenothera and Borage are cultivated as a commercial source for GLA. It would be beneficial if agronomic seed oils could be engineered to produce GLA in significant quantities by introducing a heterologous .DELTA.6-desaturase gene. It would also be beneficial if other expression products associated with fatty acid synthesis and lipid metabolism could be produced in plants at high enough levels so that commercial production of a particular expression product becomes feasible.
As disclosed in U.S. Pat. No. 5,552,306, a cyanobacterial .DELTA..sup.6 -desaturase gene has been recently isolated. Expression of this cyanobacterial gene in transgenic tobacco resulted in significant but low level GLA accumulation. (Reddy et al. 1996 Nature Biotech. 14:639-642). Applicant's copending U.S. application Ser. No. 08,366,779, discloses a .DELTA.6-desaturase gene isolated from the plant Borago officinalis and its expression in tobacco under the control of the CaMV 35S promoter. Such expression resulted in significant but low level GLA and octadecatetraenoic acid (ODTA or OTA) accumulation in seeds. Thus, a need exists for a promoter which functions in plants and which consistently directs high level expression of lipid metabolism genes in transgenic plant seeds.
Oleosins are abundant seed proteins associated with the phospholipid monolayer membrane of oil bodies. The first oleosin gene, L3, was cloned from maize by selecting clones whose in vitro translated products were recognized by an anti-L3 antibody (Vance et al. 1987 J. Biol. Chem. 262:11275-11279). Subsequently, different isoforms of oleosin genes from such different species as Brassica, soybean, carrot, pine, and Arabidopsis have been cloned (Huang, A. H. C., 1992, Ann. Reviews Plant Phys. and Plant Mol. Biol. 43:177-200; Kirik et al., 1996 Plant Mol. Biol. 31:413-417; Van Rooijen et al., 1992 Plant Mol. Biol. 18:1177-1179; Zou et al., Plant Mol. Biol. 31:429-433. Oleosin protein sequences predicted from these genes are highly conserved, especially for the central hydrophobic domain. All of these oleosins have the characteristic feature of three distinctive domains. An amphipathic domain of 40-60 amino acids is present at the N-terminus; a totally hydrophobic domain of 68-74 amino acids is located at the center; and an amphipathic .alpha.-helical domain of 33-40 amino acids is situated at the C-terminus (Huang, A. H. C. 1992).
The present invention provides 5' regulatory sequences from an oleosin gene which direct high level expression of lipid metabolism genes in transgenic plants. In accordance with the present invention, chimeric constructs comprising an oleosin 5' regulatory region operably linked to coding sequence for a lipid metabolism gene such as a .DELTA.6-desaturase gene are provided. Transgenic plants comprising the subject chimeric constructs produce levels of GLA approaching the level found in those few plant species which naturally produce GLA such as evening primrose (Oenothera biennis).